Two-year durability of REBYOTA™ (RBL), a live biotherapeutic for the prevention of recurrent Clostridioides difficile infections

Clostridioides di cile infection (CDI), the leading cause of antibioticand healthcare-associated infectious diarrhea in the United States, is associated with almost half a million infections annually [1, 2]. Dysbiosis, a perturbation in the intestinal microbiota composition characterized by reduced richness and diversity in the gut microbiome is implicated in CDI [3-5]. While antibiotics are the standard treatment for CDI [6], they o en lead to microbial dysbiosis that predisposes patients to recurrence [7]; between 20–35% of patients experience recurrent CDI (rCDI) [8], of which up to 60% experience subsequent recurrences [9]. Recurrence leads to signi cant morbidity and mortality along with substantial economic and quality of life implications that increase with each subsequent recurrence [10].


Introduction
Clostridioides di cile infection (CDI), the leading cause of antibiotic-and healthcare-associated infectious diarrhea in the United States, is associated with almost half a million infections annually [1,2].Dysbiosis, a perturbation in the intestinal microbiota composition characterized by reduced richness and diversity in the gut microbiome is implicated in CDI [3][4][5].While antibiotics are the standard treatment for CDI [6], they o en lead to microbial dysbiosis that predisposes patients to recurrence [7]; between 20-35% of patients experience recurrent CDI (rCDI) [8], of which up to 60% experience subsequent recurrences [9].Recurrence leads to signicant morbidity and mortality along with substantial economic and quality of life implications that increase with each subsequent recurrence [10].
Most CDI recurrences occur rapidly within a few weeks of antibiotic completion [11,12].However, there is a paucity of data regarding the incidence of CDI events over more than a few months.Data from the Premier Healthcare Database showed that some patients experienced up to ve or more episodes in one year [13].Given that antibiotics are not always e ective at achieving a sustained cure, additional treatments that repair the microbiome a er discontinuation of antibiotic treatment are needed for patients with rCDI.
A common clinical scenario is patients who develop rCDI due to repeated antibiotic exposure and are at risk of getting ongoing antibiotics leading to further recurrences.We discuss the representative case of a 78-year-old female with a history of depression, hypercholesterolemia, obesity, C. di cile infection a few years ago, and recurrent bacterial sinusitis for which she recently received 875/125 mg amoxicillin/clavulanate twice daily (BID) for 10 days.Her sinus symptoms improved but three days later she developed diarrhea.A stool sample for C. di cile toxin testing was positive and she was initially prescribed daxomicin 200 mg BID for 10 days but could not ll the prescription due to cost, so she was given vancomycin 125 mg four times daily (QID) for 10 days.Symptoms improved but 10 days later the diarrhea returned with moderate abdominal pain.e patient was brie y admitted to the hospital and given a vancomycin taper: 125 mg QID, followed by 125 mg BID for seven days, then one capsule daily for seven days, then one capsule every other day for 14 days.Her stools returned to normal both in frequency and consistency.One month later her diarrhea returned which was again C. di cile toxin positive.She refused daxomicin again due to cost and was prescribed another vancomycin taper and pulse regimen.She was distressed with her experience and was wondering about recurrence prevention strategies due to multiple recurrences of CDI in a short period.
As a clinician you discuss microbiota-based therapies including fecal microbiota transplantation and an available live biotherapeutic product (LBP), Fecal microbiota Live -jslm (RBL / Rebyota TM ).She is interested in long term e cacy and safety of these therapeutics.

Fecal Microbiota Transplantation (FMT)
FMT has emerged as a well-recognized strategy for the management of rCDI by restoring gut microbiota homeostasis [14].CDI treatment guidelines published by both the Infectious Diseases Society of America and the American College of Gastroenterology now endorse FMT as a recommended therapy for rCDI unresponsive a er two or more recurrences treated with antibiotics [6,15].
One multicenter retrospective study investigated the e cacy and safety of a FMT protocol that utilized a mix of fresh or frozen stool InSight obtained from patient-directed and universal donors with recognized donor selection and screening processes.
e FMT protocol achieved an 89.4% short-term cure rate (absence of CDI at ≤8 weeks), an 85.5% 6-month cure rate (absence of CDI at ≤6 months), and a 75.8% sustained cure (the absence of CDI at any timepoint a er FMT).Half of these patients reported new medical conditions or diagnoses post-FMT, but the majority were considered unlikely to be related to the treatment.Furthermore, no new serious infections developed and there was no clustering of diseases associated with dysbiosis such as obesity, hyperlipidemia, and hypertension [16].
ere are several challenges with FMT, these include lack of standardization of the product processing, burden of comprehensive pathogen screening and donor availability.ese issues led to the development of standardized biologic therapeutic products which avoided these issues.e rst live biotherapeutic product, REBYOTA TM , was approved by the US Food and Drug administration (FDA) in November 2022.

Live biotherapeutic products (LBP)
While the Centers for Disease Control and Prevention and the United States FDA consider CDI treatment success as prevention of recurrence for eight weeks a er treatment [17], data on the long-term success of CDI treatments is lacking.Determining whether these products can maintain e cacy beyond six months is important to determine whether they can provide a long-term solution to the problem of recurrence of CDI.

REBYOTA TM (RBL)
RBL is an FDA-approved, rectally administered, human-derived, broad consortium microbiota-based LBP for rCDI.A phase 3 trial of a single dose of RBL demonstrated e cacy of 70.6% at week 8 in subjects with ≥2 episodes of CDI and >90% of subjects with treatment success had sustained response through six months [18].
Long-term e cacy and safety data are available up to two years for RBL in adults (≥18 years old) with a diagnosis of rCDI and either ≥ 2 recurrences of CDI following ≥2 rounds standard-of-care antibiotic therapy or ≥2 episodes of severe CDI requiring hospitalization [19,20].
In the PUNCH CD Open Label Phase 2 trial, subjects who received two doses of RBL demonstrated a 78.9% treatment success rate at 56 days post-treatment compared to 30.7% for the historical control group (p < 0.0001).Among the RBL responders, 97% remained CDI-free at six months while 95% and 91% remained occurrence-free at one and two years, respectively [19].Post-hoc sub-analyses demonstrated sustained treatment response through six months, one year, and two years a er treatment for 98%, 98%, and 94% of patients <65 years of age, respectively, and 97%, 93%, and 88% of patients ≥65 years of age.Similarly, 98%, 96%, and96% of patients with 2 or 3 prior CDI episodes, respectively, and 97%, 94%, and 86% of patients with ≥4 episodes remained recurrence-free at six months, one year, and two years.Similar sustained response rates were also demonstrated in patients categorized by gender and race [21].
roughout the study period, most TEAEs were mild to moderate (84%), primarily related to pre-existing conditions (58%), and were experienced during the rst four weeks a er treatment (60%).e most common adverse events were gastrointestinal in nature with diarrhea reported in 30% of subjects.Importantly, there were no reported infections caused by pathogens traceable to RBL.While 13% of subjects experienced urinary tract infections, none were deemed related to RBL or the enema procedure.Only two subjects reported serious adverse events assessed as possibly related to RBL, including ileus, leukocytosis, pyrexia, atrial brillation, coincident with severe CDI eventually leading to death in one subject and three CDI episodes (one severe) in the other subject; but these were also deemed related to pre-existing conditions and CDI [19].
In the PUNCH CD2 randomized, placebo-controlled Phase 2b trial, subjects were randomized 1:1:1 to receive two doses of RBL, two doses of placebo, or one dose of RBL followed by one dose of placebo.e primary e cacy endpoint was treatment success a er two doses of RBX2660 compared with two doses of placebo.While the prespeci ed intent-to-treat e cacy analysis was not statistically signi cant [22], in the nal per-protocol population analysis, treatment success was achieved in 87.5% of subjects who received one dose of RBL followed by one dose of placebo versus 58.1% in the placebo-only group (p = 0.017).
ere was no signi cant di erence in treatment response between the two doses of RBL (75%) and placebo (p = 0.17) [20].Among the RBL responders in the two-dose group, 100% remained CDI-free at three months whereas 91.7% of the two dose RBL group remained recurrence-free at six months and 87.5% remained recurrence-free at both one and two years.e one dose RBL group achieved a more sustained clinical response as 95.7% remained recurrence-free at at six months, one year, and two years (data on le, Ferring Pharmaceuticals).
e safety pro le of RBL, whether delivered as one or two doses, was like the placebo group; most (87.6%) of the TEAEs were mild or moderate in severity, and 32.5% were primarily related to gastrointestinal disorders.ree (2.3%) serious adverse events (constipation, recurrent acute myeloid leukemia, abdominal pain) were reported as possibly related to RBL.
ere were no reports of bacteremia or fungemia over the two-year period [20].
RBL has been shown to e ectively restore dysbiosis within the gut microbiome.A er administration, the gut microbiome in RBL responders becomes more similar to the unperturbed microbiome of RBL, and hence more closely resembles the microbiome of healthy patients.Microbiota changes occur within seven days of treatment and last for six months to two years; treatment not only increases alpha diversity, but it also signi cantly increases Bacteroidia and Clostridia, which are restored to predominance, and decreases Gammaproteobacteria, Bacilli, and Erysipelotrichia  [19, 23-25 RBL restores bile acid metabolism towards healthier bile acid pro les characterized by higher secondary:primary bile acid ratios.
A bile acid composition enriched with secondary bile acids is normally seen in the healthy gut whereas enrichment with primary bile acids, as seen in baseline in Figure 1, is associated with dysbiosis and an increased risk of CDI recurrence [26,27].To quantify mean changes to bile compositions induced by RBL treatment, bile acids were categorized into primary conjugated, primary deconjugated, secondary deconjugated, and secondary conjugated.A er RBL administration, Figure 1 illustrates a signi cant drop in primary bile acids concurrent with increased secondary bile acids that increased over time plateauing a er eight weeks and remaining sustained throughout two years a er treatment [26].e shi s in secondary versus primary bile acids support the continued e cacy over two years.

Case resolution
Upon discussion of options, she opted for fecal microbiota Livejslm (RBL /REBYOTA TM ) due to the non-invasive nature of the product and the long-term safety and e cacy data available.She underwent rectal administration a er a wash out period of 48 hours a er vancomycin and did not experience any di culty with the administration and had no adverse reactions.

Conclusions
Microbiota-directed approaches for preventing CDI recurrences can provide both short-and long-term bene t. e data thus far suggests that the rst FDA-approved LBP RBL can prevent rCDI in initial responders for up to two years a er treatment administration.
Clinical reduction of CDI recurrence is correlated with restorative microbiome changes, including signi cantly increased alpha diversity as well as an increased abundance of Bacteroidia and Firmicutes including Clostridia-class bacteria and a decreased abundance of Gammaproteobacteria and Bacilli.
Healthier bile acid pro les, including signi cantly reduced primary bile acids and restored secondary bile acids are also correlated with clinical reduction of CDI recurrence.

Restoration of microbial diversity and higher secondary:primary bile acid ratios are associated with a continued CDI-free state in RBL-treated subjects
].The microbiome is dysbiotic based on relative abundance of Gammaproteobacteria vs Firmicutes and Bacteroidetes Ferdyan N, Srinivasan K, et al.Human Fecal Bile Acid Analysis after Investigational Microbiota-Based Live Biotherapeutic Delivery for Recurrent Clostridioides difficile Infection.Microorganisms.2023;11(1):135.*clinicalefficacy at time point